The yeast deletion collections comprise >21,000 mutant strains that carry precise start-to-stop deletions of ∼6000 open reading frames. This collection includes heterozygous and homozygous diploids, ...and haploids of both MAT A: and MATα mating types. The yeast deletion collection, or yeast knockout (YKO) set, represents the first and only complete, systematically constructed deletion collection available for any organism. Conceived during the Saccharomyces cerevisiae sequencing project, work on the project began in 1998 and was completed in 2002. The YKO strains have been used in numerous laboratories in >1000 genome-wide screens. This landmark genome project has inspired development of numerous genome-wide technologies in organisms from yeast to man. Notable spinoff technologies include synthetic genetic array and HIPHOP chemogenomics. In this retrospective, we briefly describe the yeast deletion project and some of its most noteworthy biological contributions and the impact that these collections have had on the yeast research community and on genomics in general.
Pharmacogenomics can enhance patient care by enabling treatments tailored to genetic make-up and lowering risk of serious adverse events. As of June 2019, there are 132 pharmacogenomic dosing ...guidelines for 99 drugs and pharmacogenomic information is included in 309 medication labels. Recently, the technology for identifying individual-specific genetic variants (genotyping) has become more accessible. Next generation sequencing (NGS) is a cost-effective option for genotyping patients at many pharmacogenomic loci simultaneously, and guidelines for implementation of these data are available from organizations such as the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG). NGS and related technologies are increasing knowledge in the research sphere, yet rates of genomic literacy remain low, resulting in a widening gap in knowledge translation to the patient. Multidisciplinary teams-including physicians, nurses, genetic counsellors, and pharmacists-will need to combine their expertise to deliver optimal pharmacogenomically-informed care.
Abstract
Using a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two ...related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.
Chemogenomic profiling is a powerful approach for understanding the genome-wide cellular response to small molecules. First developed in Saccharomyces cerevisiae, chemogenomic screens provide direct, ...unbiased identification of drug target candidates as well as genes required for drug resistance. While many laboratories have performed chemogenomic fitness assays, few have been assessed for reproducibility and accuracy. Here we analyze the two largest independent yeast chemogenomic datasets comprising over 35 million gene-drug interactions and more than 6000 unique chemogenomic profiles; the first from our own academic laboratory (HIPLAB) and the second from the Novartis Institute of Biomedical Research (NIBR).
Despite substantial differences in experimental and analytical pipelines, the combined datasets revealed robust chemogenomic response signatures, characterized by gene signatures, enrichment for biological processes and mechanisms of drug action. We previously reported that the cellular response to small molecules is limited and can be described by a network of 45 chemogenomic signatures. In the present study, we show that the majority of these signatures (66%) are also found in the companion dataset, providing further support for their biological relevance as conserved systems-level, small molecule response systems.
Our results demonstrate the robustness of chemogenomic fitness profiling in yeast, while offering guidelines for performing other high-dimensional comparisons including parallel CRISPR screens in mammalian cells.
Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens ...and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic's mode of action against fungal cell wall biosynthesis. Both classical- and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.
A series of reports over the last few years have indicated that a much larger portion of the mammalian genome is transcribed than can be accounted for by currently annotated genes, but the quantity ...and nature of these additional transcripts remains unclear. Here, we have used data from single- and paired-end RNA-Seq and tiling arrays to assess the quantity and composition of transcripts in PolyA+ RNA from human and mouse tissues. Relative to tiling arrays, RNA-Seq identifies many fewer transcribed regions ("seqfrags") outside known exons and ncRNAs. Most nonexonic seqfrags are in introns, raising the possibility that they are fragments of pre-mRNAs. The chromosomal locations of the majority of intergenic seqfrags in RNA-Seq data are near known genes, consistent with alternative cleavage and polyadenylation site usage, promoter- and terminator-associated transcripts, or new alternative exons; indeed, reads that bridge splice sites identified 4,544 new exons, affecting 3,554 genes. Most of the remaining seqfrags correspond to either single reads that display characteristics of random sampling from a low-level background or several thousand small transcripts (median length = 111 bp) present at higher levels, which also tend to display sequence conservation and originate from regions with open chromatin. We conclude that, while there are bona fide new intergenic transcripts, their number and abundance is generally low in comparison to known exons, and the genome is not as pervasively transcribed as previously reported.
Insulin receptor (Insr) protein is present at higher levels in pancreatic β-cells than in most other tissues, but the consequences of β-cell insulin resistance remain enigmatic. Here, we use an Ins1
...knock-in allele to delete Insr specifically in β-cells of both female and male mice. We compare experimental mice to Ins1
-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined β-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout β-cells from female, but not male mice, whereas only male βInsr
islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female βInsr
and βInsr
mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter β-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include β-cell insulin resistance, which predicts that β-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female βInsr
and βInsr
mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of β-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that β-cell insulin resistance in the form of reduced β-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.
The history of yeast Saccharomyces cerevisiae, aka brewer's or baker's yeast, is intertwined with our own. Initially domesticated 8,000 years ago to provide sustenance to our ancestors, for the past ...150 years, yeast has served as a model research subject and a platform for technology. In this review, we highlight many ways in which yeast has served to catalyze the fields of functional genomics, genome editing, gene-environment interaction investigation, proteomics, and bioinformatics-emphasizing how yeast has served as a catalyst for innovation. Several possible futures for this model organism in synthetic biology, drug personalization, and multi-omics research are also presented.
In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised ...World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.
The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components ...can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk of age-related disease in mammals. We compared Ins2+/− mice to Ins2+/+ littermate controls, on a genetically stable Ins1 null background. Proteomic and transcriptomic analyses of livers from 25-week-old mice suggested potential for healthier aging and altered insulin sensitivity in Ins2+/− mice. Halving Ins2 lowered circulating insulin by 25%–34% in aged female mice, without altering Igf1 or circulating Igf1. Remarkably, decreased insulin led to lower fasting glucose and improved insulin sensitivity in aged mice. Moreover, lowered insulin caused significant lifespan extension, observed across two diverse diets. Our study indicates that elevated insulin contributes to age-dependent insulin resistance and that limiting basal insulin levels can extend lifespan.
Display omitted
•Mechanistic correlates of healthy aging were identified in 25-week-old Ins2+/− mice•Mild life-long reduction in insulin levels improves insulin sensitivity with aging•Reducing circulating insulin by ∼25% extends lifespan in mice, across diverse diets
In a study examining the life-long effects of lowering insulin levels in mice, Templeman et al. provide evidence that elevated insulin levels contribute to age-dependent insulin resistance. Moreover, they show that slightly reducing circulating insulin is sufficient to extend mammalian lifespan, independently of Igf1.
PDF
